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1.  Structural and functional studies of Igαβ and its assembly with the B cell antigen receptor 
The B cell antigen receptor (BCR) plays an essential role in all phases of B cell development. Here, we show the extracellular domains of murine and human Igβ form an I-set immunoglobulin-like structure with an inter-chain disulfide between cysteines on their G-strands. Structural and sequence analysis suggests that Igα displays similar fold as Igβ. An Igαβ heterodimer model was generated based on the unique disulfide bonded Igβ dimer. Solution binding studies showed that the extracellular domains of Igαβ preferentially recognized the constant region of BCR with the μ-chain specificity, suggesting a role for Igαβ to enhance the BCRμ-chain signaling. Cluster mutations on Igα, Igβ and mIgM based on the structural model identified distinct area of potential contacts involving charged residues on both subunits of the co-receptor and the Cμ4 domain of mIgM. These studies provide the first structural model for understanding BCR function.
PMCID: PMC2921123  PMID: 20696394
2.  A case of structure determination using pseudosymmetry 
When properly applied, pseudosymmetry can be used to improve crystallographic phases through averaging and to facilitate crystal structure determination.
Here, a case is presented of an unusual structure determination which was facilitated by the use of pseudosymmetry. Group A streptococcus uses cysteine protease Mac-1 (also known as IdeS) to evade the host immune system. Native Mac-1 was crystallized in the orthorhombic space group P21212. Surprisingly, crystals of the inactive C94A mutant of Mac-1 displayed monoclinic symmetry with space group P21, despite the use of native orthorhombic Mac-1 microcrystals for seeding. Attempts to solve the structure of the C94A mutant by MAD phasing in the monoclinic space group did not produce an interpretable map. The native Patterson map of the C94A mutant showed two strong peaks along the (1 0 1) diagonal, indicating possible translational pseudosymmetry in space group P21. Interestingly, one-third of the monoclinic reflections obeyed pseudo-orthorhombic P21212 symmetry similar to that of the wild-type crystals and could be indexed and processed in this space group. The pseudo-orthorhombic and monoclinic unit cells were related by the following vector operations: a m = b o − c o, b m = a o and c m = −2c o − b o. The pseudo-orthorhombic subset of data produced good SAD phases, leading to structure determination with one monomer in the asymmetric unit. Subsequently, the structure of the Mac-1 mutant in the monoclinic form was determined by molecular replacement, which showed six molecules forming three translationally related dimers aligned along the (1 0 1) diagonal. Knowing the geometric relationship between the pseudo-orthorhombic and the monoclinic unit cells, all six molecules can be generated in the monoclinic unit cell directly without the use of molecular replacement. The current case provides a successful example of the use of pseudosymmetry as a powerful phase-averaging method for structure determination by anomalous diffraction techniques. In particular, a structure can be solved in a higher pseudosymmetry subcell in which an NCS operator becomes a crystallographic operator. The geometrical relationships between the subcell and parental cell can be used to generate a complete molecular representation of the parental asymmetric unit for refinement.
PMCID: PMC2789005  PMID: 19966420
pseudosymmetry; structure determination; cysteine proteases; Mac-1
3.  A rational approach to heavy-atom derivative screening 
In order to overcome the difficulties associated with the ‘classical’ heavy-atom derivatization procedure, an attempt has been made to develop a rational crystal-free heavy-atom-derivative screening method and a quick-soak derivatization procedure which allows heavy-atom compound identification.
Despite the development in recent times of a range of techniques for phasing macromolecules, the conventional heavy-atom derivatization method still plays a significant role in protein structure determination. However, this method has become less popular in modern high-throughput oriented crystallography, mostly owing to its trial-and-error nature, which often results in lengthy empirical searches requiring large numbers of well diffracting crystals. In addition, the phasing power of heavy-atom derivatives is often compromised by lack of isomorphism or even loss of diffraction. In order to overcome the difficulties associated with the ‘classical’ heavy-atom derivatization procedure, an attempt has been made to develop a rational crystal-free heavy-atom derivative-screening method and a quick-soak derivatization procedure which allows heavy-atom compound identification. The method includes three basic steps: (i) the selection of likely reactive compounds for a given protein and specific crystallization conditions based on pre-defined heavy-atom com­pound reactivity profiles, (ii) screening of the chosen heavy-atom compounds for their ability to form protein adducts using mass spectrometry and (iii) derivatization of crystals with selected heavy-metal compounds using the quick-soak method to maximize diffraction quality and minimize non-isomorphism. Overall, this system streamlines the pro­cess of heavy-atom compound identification and minimizes the problem of non-isomorphism in phasing.
PMCID: PMC2852299  PMID: 20382988
heavy-atom derivativization; heavy-atom screening; phasing; structure determination

Results 1-3 (3)